Bioactive glass preparation and use

ABSTRACT

A process of preparing a glass comprising: (a) heating a mixture of precursor chemicals to a melt temperature to form a melt, the melt being characterized in that quenching the melt at or above a threshold temperature results in a spinodal phase seperation, and quenching the melt below the threshold temperature results in a droplet phase seperation; and (b) quenching the melt at or above the threshold temperature in a preheated mold to form the glass composition having the spinodal phase seperation.

REFERENCE TO RELATED APPLICATION

This application is related to U.S. Provisional Application No.61/896,227 filed Oct. 28, 2013 and hereby incorporated by reference.

FIELD OF INVENTION

The present invention relates generally to a bioactive glass and, morespecifically, to a bioactive glass prepared to favor spinodal phaseseperation.

BACKGROUND

With the advent of tissue engineering, the scientific and medicalcommunities are rapidly shifting from an emphasis on tissue replacementto tissue regeneration. Bioactive glass has been recognized for itsremarkable biocompatibility and its potential has recently been growingas a highly promising material for hard tissue repair and regeneration.

Manufacturing methods and post-fabrication treatment of 45S5 glass(45SiO₂-24.5Na₂O-24.5CaO-6P₂O₅ by wt. %), which is a well-knownbioglass, are shown to have a significant impact on its biologicalresponse. Many studies have been devoted to understand the effect ofdevitrification of this bioactive glass (BG) on its physical propertiesas well as biological performance such as its ability to promote bonegrowth and regeneration. The BG-derived glass-ceramics (crystalline orsemi-crystalline) or bioscaffolds prepared by the sintering of theirpowder exhibit suitable mechanical properties along with broaderengineering possibilities. At the same time, the glass-ceramics,compared to BG, show different solubility in body fluid and possibly theprotein adsorption profile (protein amount and conformation can dependon surface morphology)—a key factor influencing cells attachment. Acommon argument is that the phosphorous distribution changes uponcrystallization of a glass, causing phosphorous dissolution profile tochange, which ultimately affects the distribution of binding sites forproteins and the time to form the hydroxycarbonate apatite (HCA) layerneeded for tissue integration.

Although the phosphorous dissolution profile may affect bioactivity,Applicants, recognize that nanostructure of the glass also plays asignificant role in promoting bioactivity, and have identified a need toinvestigate and establish the desirable nanostructure of glass and itseffects on bioactivity. The present invention fulfills this need amongothers.

SUMMARY OF INVENTION

The following presents a simplified summary of the invention in order toprovide a basic understanding of some aspects of the invention. Thissummary is not an extensive overview of the invention. It is notintended to identify key/critical elements of the invention or todelineate the scope of the invention. Its sole purpose is to presentsome concepts of the invention in a simplified form as a prelude to themore detailed description that is presented later.

Despite the progress in the applications of 45S5 BG and correspondingceramics, the fact that this composition belongs to the immiscibilitydomain of corresponding phase diagram appears to be largelyunappreciated. The origin of such phase separation pertains to apositive enthalpy of mixing, which drives a glass-forming liquid toseparate spontaneously into two compositionally distinct phases. It is aprocess concurrent to the maximization of entropy, which usually resultsin a homogeneous mixing of the constituents in glass. Liquid-liquidimmiscibility can lead to droplet-like or spinodal-type phase separationin the glassy state. Droplet phase separation is characterized by asharp boundary between the composition of droplets and rest of thematrix, while in spinodal decomposition, the phase separated systemconsists of two interpenetrating networks of diffuse compositionalboundary between the two immiscible phases. In the latter case, thecompositions of two phases evolve simultaneously as the degree of phaseseparation increases and the boundary between phases becomes moredistinct.

One aspect of the present invention is Applicants' recognition that 45S5glass/ceramic product can be phase-separated at the nanoscale, and canhave unique properties depending on the quenching rate orpost-fabrication treatment (annealing, etc.). Specifically, differentdegrees of spinodal phase separation with two interpenetrating phasescan be obtained by varying the melt temperature within 1400-1600° C.range and casting into preheated molds. Additionally, Applicantsdiscovered that glass compositions have a threshold temperature forquenching, above which results in glass with a spinodal phaseseparation, and below which results in the glass with a droplet phaseseparation. For example, quenching a meld of 45S5 at 1380° C. tends toproduce spinodal phase separation, while quenching at 1370° C. tends toproduce droplet phase separation. Thus, for 45S5, the thresholdtemperature lies between 1370 and 1380° C.

On the other hand, conventional glass is manufactured to producehomogeneous, defect free solid at the lowest cost, which implies workingat the lowest possible temperature and using minimum number ofprocessing steps. For example, the conventional 45S5 glass may be meltedat temperatures below 1400° C., cast in a mold at ambient temperatureand then annealed to relieve stresses. Applicants recognize that in thisconventional process, the nucleation and growth mechanism, whichproduces isolated droplets in an otherwise continuous phase, overrulesspinodal type phase separation when melt is cast from 1250-1400° C. intounheated molds. In contrast, the glass produced from the method of thepresent invention has interconnected phases in a spinodal typemicrostructure (unlike commonly prepared glass of same composition),which may be single phase or have phase separation as droplets in acontinuous matrix. Thus, the nanostructure of 45S or any other glass,can be controlled by varying the temperature at which the melt ismaintained before casting, the temperature of the mold and subsequentcooling routine.

Another aspect of the present invention is the examination of theclassic melt-quenched 45S5 glass composition with signatures ofdroplet-like or spinodal phase-separation at the nanoscale, as well ascorresponding ceramics prepared by the devitrification of these parentglasses. In this respect, little is known about the influence ofnanoscale phase-separation on the performance of scaffolds made of 45S5BG and/or corresponding glass-ceramics. For instance, the physicalproperties of 45S5 glass phase-separated at the nanoscale, and theinfluence of this phase separation on the crystallization kinetics,glass-ceramics formation, HCA layer formation, or ionicdissolution/leaching rates is generally unexplored.

Applicants discovered that the type of phase separation (such asspinodal vs. droplet-like) has a pronounced effect on a variety ofcharacterisitcs, including the activation energy of viscous flow andcrystallization, the onset temperature of crystallization, and the voidsize distribution at the nanoscale. Furthermore, Applicants examined thecellular ability to detect differences in nanostructure on 45S5bioactive glass samples. Applicants have discovered that a 45S glasscomprising spinodal type nanostructure is biomedically superior to the45S glass comprising droplet type nanostructure. Specifically,Applicants found cells showed a preference to the spinodal phaseseperation as opposed to the droplette distribution, suggesting thatcells are somehow sensing the details of the morphology of thesubstrates that are about 1000 times smaller than the cells themselves.The advantage of spinodal nanostructure applies not only to 45S5, butalso to other silicate compositions as well, such as those derived fromstandard 45S composition for instance.

Therefore, Applicants not only realized the importance of the spinodaland droplette nanostruture of glass and identified a method of producingeach, but also recognized that phase separation nanostruture hassignificant effects on the characterisitics of the glass, including theability of cells to attach and prolifferate on the glass material.

One aspect of the invention is a method of preparing a glass compositionby quenching a glass melt at or above a threshold temperature to promotea spinodal phase separation. In one embodiment, the method comprises:(a) heating a mixture of chemicals to a melt temperature to form a melt,the melt being characterized in that quenching the melt at or above athreshold temperature results in a spinodal phase separation, andquenching the melt below the threshold temperature results in a dropletphase separation; and (b) quenching the melt at or above the thresholdtemperature in a preheated mold to form the glass composition having thespinodal phase separation.

Another aspect of the invention is a glass composition prepared byquenching a glass melt at or above a threshold temperature to promote aspinodal phase separation. In one embodiment, the glass composition isprepared by the process comprising: (a) heating a mixture of chemicalsto a melt temperature to form a melt, the melt being characterized inthat quenching the melt at or above a threshold temperature results in aspinodal phase separation, and quenching the melt below the thresholdtemperature results in a droplet phase separation; and (b) quenching themelt at or above the threshold temperature in a preheated mold to formthe glass composition having the spinodal phase separation.

Yet another aspect of the invention is a method of using the glasscomposition to promote cellular attachment and proliferation. In oneembodiment, the method comprises: (a) disposing the glass composition ina cellular environment, the glass being prepared by a process comprisingat least: (i) heating a mixture of chemicals to a melt temperature toform a melt, the melt being characterized in that quenching the melt ator above a threshold temperature results in a spinodal phase seperation,and quenching the melt below the threshold temperature results in adroplet phase seperation; and (ii) quenching the melt at or above thethreshold temperature in a preheated mold to form the glass compositionhaving the spinodal phase seperation; and (c) facilitating cellularattachment and proliferation on the glass composition.

BRIEF DESCRIPTION OF FIGURES

FIG. 1( a) shows 45S5 glasses phase separated to different degrees.

FIGS. 1( b) and (c) show SEMs of droplet-like phase separation andspinodal phase separation, respectively.

FIG. 2 shows FT-IR spectra of droplet-like and spinodally phaseseparated 45S5 glasses, with spectrum of fused silica shown forcomparison.

FIGS. 3( a) and (b) show XRD spectra of spinodally and droplet-likephase separated 45S5 glasses, and obtained ceramics, respectively.

FIG. 4( a)-(d) show typical DSC heating curves for spinodally (a, b) anddroplet-like (c, d) phase separated 45S5 glasses measured for the bulk(a, c) and <32 μm powder (b, d) samples.

FIGS. 5( a) and (b) show DSC heating curves for spinodally anddroplet-like phase separated 45S5 glasses, respectively, measured fordifferent particle size at 5 K/min heating rate.

FIG. 6 shows Ozawa plots for the estimation of the crystallizationactivation energy for spinodally phase-separated glass of differentparticle size.

FIG. 7 shows Ozawa plots for the estimation of the crystallizationactivation energy for droplet-like phase-separated glass of differentparticle size.

FIG. 8 shows a typical polycrystal formed at the surface of 45S5 sample.

FIGS. 9( a) and (b) show Z(α) functions for spinodally and droplet-likephase separated 45S5 glasses, respectively, wherein open symbolscorrespond to bulk samples, full symbols stand for <32 μm size powder.

FIGS. 10( a) and (b) show positron annihilation lifetime (PAL) spectraobtained for spinodally and droplet-like phase-separated 45S5 glassesand ceramics, respectively, with example of fitting components for glasssamples.

FIG. 11 is a schematic of one embodiment of the method of the presentinvention.

FIGS. 12( a) and (b) illustrate droplet and spinodal nanostructures,respectively, formed by quenching from varying temperatures.

FIGS. 13( a) and (b) are SEM photos showing droplet and spinodalnanostructures, respectively.

FIGS. 14( i)-(iv) illustrate nanostructure affecting cellular densityand adhesion.

FIGS. 15 (i)-(iv) shows results confirming that cells respond todifferences in the nanostructure of 45S5 bioglass.

DETAILED DESCRIPTION

In one embodiment, the present invention involves a process of preparinga glass comprising heating a mixture of chemicals to a melt temperatureto form a melt, the melt being characterized in that quenching the meltat or above a threshold temperature results in a spinodal phaseseperation, and quenching the melt below the threshold temperatureresults in a droplet phase seperation; and quenching the melt at orabove the threshold temperature to form the glass composition having thespinodal phase seperation.

The mixture may be any mixture for producing a glass having spinodalphase separation. Suitable mixtures include, for example, the mixture ofhigh purity (99.99% or better) CaCO₃, Na₂CO₃, silicon dioxide SiO₂ andcalcium phosphate tribasic Ca₅OH(PO₄)₃ powders, for 45S5 glass (45 wt. %SiO₂-24.5 wt. % Na₂O-24.5 wt. % CaO-6 wt. % P₂O₅), and other bioactivesilicate glasses that are prone to phase separation, for example, thederivatives of 45S5 composition doped with the oxides of silver, zinc,strontium, boron, titanium, etc., or different ratios of theconstituents of 45S5 composition.

The melt temperature can vary based on the mixture and other processparameters including the temperature of the mold and quenchingtemperature. For example, suitable results have been obtained for 45S5having a melt temperature of about 1400 to about 1600° C. As mentionedabove, this melt temperature is generally higher than that used toprepare conventional 45S5. Applicants recognize that in the conventionalprocess, the nucleation and growth mechanism, which produces isolateddroplets in an otherwise continuous phase, overrules spinodal type phaseseparation when the melt is cast, for example, from 1250-1400° C. intounheated molds. In contrast, the glass produced from the method of thepresent invention has interconnected phases in a spinodal typedistribution. A different degree of spinodal phase separation with twointerpenetrating phases can be obtained by varying the melt temperaturewithin 1400-1550° C. range and casting into preheated molds. In oneembodiment, the melt temperature is about 1450 to about 1550° C., and,in a more particular embodiment, the melt temperature is about 1550° C.

The threshold temperature will vary depending on the composition of themixture, among other variables. For example, suitable results have beenobtained in which the threshold temperature is no less than about 86% ofthe melt temperature. In a more particular embodiment, the thresholdtemperature is no less than about 88.5% of the melt temperature, and ina more particular embodiment, the threshold temperature is no less thanabout 89% of the melt temperature. For example, in one embodiment, inwhich the molds are heated above 100° C. and a 45S5 mixture is used, thethreshold temperature is no less than about 1375° C. In a moreparticular embodiment, the threshold temperature ranges from 1375 toabout 1550° C., and in a still more particular embodiment, the thresholdtemperature ranges from about 1375 to about 1380° C.

In one embodiment, the quenching comprises pouring the melt into apreheated mold. Again, the degree to which the mold is preheated willdepend on the mixture and other heating parameters, although suitableresults have been achieved with a mold preheated above 100° C. In a moreparticular embodiment, the mold is preheated to about 100 to about 300°C.

It should be understood that the process of the preparing the spinodalphase separated glass may involve other steps which are well known inthe art. For example, in one embodiment, the mixture is calcined beforeforming the melt. Suitable results have been achieved when calcining atabout 800° C. for 4-7 hours.

Additionally, it may be preferable to cool the melt from its melttemperature prior to quenching. For example, in one embodiment, the meltis allowed to cool gradually from 1550° C. before being quenched. In oneparticular embodiment, the melt is allowed to cool to 1380° C. before itreaches the threshold temperature.

Another aspect of the invention is the product made from the processdescribed above.

Still another aspect of the invention is a method of using the glasscomposition to promote cellular attachment and proliferation. Asmentioned above, an aspect of the invention is the recognition anddiscovery that the nanoscale structure of glass is important for itsbiomedical performance. In particular, Applicants have found that the invitro performance of glass is superior when its structure comprises ofinterconnected spinodally phase separated nanostructure as opposed tothe same glass with isolated droplets in a matrix. Cells respond morefavorably to the morphology and composition of the phases in the formerstructure than in the latter structure. Applicants note that, within thebroad classification of spinodal or droplet type nanostructures,significant variations of the distribution of two phases may also impactthe cell response. In other words, the conditions of glass fabricationmay be further optimized for improving the cell response, but the basicpremise of superior performance of the spinodal nanostructure willremain. This advantage of spinodal nanostructure applies to othersilicate compositions as well, such as those derived from standard 45S5composition for instance.

In one embodiment, the method comprises disposing the glass compositionmade from the process described above in a cellular environment, andthen facilitating cellular attachment and proliferation on the glasscomposition. The cellular environment may be any fluid environmenthaving living cells. Such an environment may be, for example, in a Petridish or in an animal body. Facilitating attachment and proliferationmeans broadly maintaining the environment to enable the cells to livefor a significant period. Such cellular environments and conditions formaintaining them are well known in the art.

Example 1

The following example demonstrates the different phase separations thatcan be formed depending on whether the melt is quenched at or above thethreshold temperature, or below the threshold temperature. Further,these examples illustrate the different properties and influences thedifferent phase separations have. Note these results are published inGolovchak et al. Influence of phase separation on the devitrification of45S5 bioglass, Acta Biomater (2014),http://dx.doi.org/10.1016/j.actbio.2014.07.024 hereby incorporated byreference. Note that these are just examples, and that, one may findother combination of process parameters to obtain the two types ofnanostructures.

Referring to FIG. 11, a schematic of one embodiment of the method ofpreparing the glass composition of the present invention is shown.Specifically, a 45S5 glass (45SiO₂-24.5Na₂O-24.5CaO-6P₂O₅ by wt. %) wassynthesized using melt-quenching and casting in stainless steel molds.High purity (99.99% or better) carbonates CaCO₃, Na₂CO₃, silicon dioxideSiO₂ and calcium phosphate tribasic Ca₅OH(PO₄)₃ powders were used as rawprecursors, and melted in a Pt crucible. The powder of a given samplewas obtained by ball milling (˜1 hour) of bulk pieces followed bysieving through a collection of 5 sieves (500 μm, 300 μm, 150 μm, 75 μm,32 μm) to separate particles by size.

The mixture of starting precursor powders was calcined at 800° C. for 6hours and then slowly (2 K/min) heated till the maximum melt temperatureof 1550° C., where it was maintained at least 3 hours to homogenize themelt. To induce spinodal phase decomposition the melt was quenched from1550° C. into preheated (200-400° C.) molds. Different degrees ofspinodal phase separation with two interpenetrating phases areobtainable by varying the melt temperature within 1400-1550° C. rangeand casting into preheated molds. The nucleation and growth mechanism,which produces isolated droplets in an otherwise continuous phase,overrules spinodal type phase separation when melt is cast from1250-1400° C. into unheated molds. Thus the nanostructure of 45S glass,which can be controlled by varying the temperature at which the melt ismaintained before casting, the temperature of the mold and subsequentcooling routine.

For comparison a commercial 45S5 Bioglass was used, which had pronounceddroplet-like phase separation. The two types of phase-separation (i.e.,spinodal and drop-like) arise from the difference in the melttemperature just prior to casting, and the quench rate duringsolidification.

The 45S5 glasses were transformed into glass-ceramics by heating in twosteps—annealing of the material at 650-670 C. for 3 hours to inducecrystal nucleation, followed by another heat treatment at ˜730-750° C.for 6 hours to facilitate their growth.

The average composition of the prepared glasses was checked with X-rayphotoelectron spectroscopy (XPS). No significant difference was foundbetween the droplet-like and spinodally phase separated glasses as setforth in Table 1.

TABLE 1 Composition of the investigated spinodally and droplet-likephase separated 45S5 glasses and derived ceramics, determined from XPSdata. Composition, at. % Sample O Si Ca Na P Theoretical 55.2 16.3 9.517.2 1.8 Spinodally phase-separated glass 50.1 18.9 8.8 20.5 1.6Ceramics out of spinocally phase- 49.4 19.3 9.4 19.9 1.9 separated glassDroplet-like phase-separated glass 49.8 17.9 9.0 21.3 1.9 Ceramics outof droplet-like phase- 50.7 19.0 8.8 19.4 2.1 separated glass

The XPS spectra were recorded in a normal emission mode on samplesurfaces freshly fractured inside the ultra-high vacuum of the ScientaESCA-300 spectrometer using monochromatic Al Kα X-rays (1486.6 eV). TheXPS data consisted of survey scans over the entire binding energy (BE)range and selected scans over the core-level photoelectron peaks ofinterest. The surface charging from photoelectron emission wasneutralized using a low energy (<10 eV) electron flood gun. Theexperimental positions of core levels for all of the investigatedsamples were adjusted by referencing to the is core level peak ofadventitious carbon at 284.6 eV. Data analysis of the XPS spectra wasconducted using the standard CASA-XPS software package.

Positron annihilation lifetime (PAL) spectra were recorded with the fastcoincidence system of 230 ps resolution (FWHM of a single Gaussian,determined by measuring 60Co isotope) at the temperature, T=22° C. andrelative humidity, RH=35%. Each PAL spectrum was measured with a channelwidth of 6.15 ps (total number of channels 8000) and contained 3·10⁶coincidences in total. Isotope ²²Na (activity ˜50 kBq) was used assource of positrons (prepared from aqueous solution of ²²NaCl, wrappedwith Kapton® foil of 12 μm thickness and sealed), which was sandwichedbetween two identical samples. All the PAL spectra of the investigatedsamples (dried at 120° C. for 4 hours in vacuum before the measurements)were decomposed into four discrete exponentialss(t)=Σ(I_(i)|τ_(i))exp(−t/τ_(i)) with average positron lifetime τ_(i)and intensity I_(i) of i^(th) positron decay component (i=1 to 4) usingstandard LT 9.0 program. The additional peaks into the envelope offitted curve were added only if they significantly improved goodness ofthe fit. The uncertainties in the determination of lifetimes (τ_(i)) andcorresponding intensities (I_(i)) were ±0.005-0.5 ns (increasing withincreasing τ_(i)) and ±0.2-1%, respectively.

FT-IR measurements on samples polished to high optical quality wereperformed in a reflection mode (nearly normal incident angle), usingVarian 7000 e spectrometer.

Rigaku “MiniFlex II” diffractometer was used for X-ray powderdiffraction (XRD) studies. The XRD patterns were recorded within 15-60°angular range, 0.02° scan step and 1 s integration time.

DSC data were acquired for bulk and powder samples using NETZSCH 404/3Fmicrocalorimeter pre-calibrated with a set of standard elements. DSCcurves were recorded in a nitrogen atmosphere with 2, 5, 10, 15 and 20K/min heating rates. Three independent DSC measurements were performedin each case to confirm the reproducibility of the obtained results. RawDSC data were processed using NETZSCH PC software package.

Spinodally phase-separated 45S5 glass appears slightly purple in thereflected light in comparison to the glass with droplet-like phaseseparation (FIG. 1 a). This color originates from the scattering oflight caused by the co-existence of two interconnected phases with closerefractive indexes; UV-VIS spectroscopy testified that no characteristiccolor lines were present in UV-VIS absorption spectra. High-resolutionSEM images in FIG. 1 b,c show the difference in the structure of thecolorless and purple tinged glasses at the nanoscale. Two interconnectedphases can be clearly seen in FIG. 1 c, which is characteristic ofspinodal type phase decomposition, whereas fingerprints of droplet-likephase separation with sharp compositional boundaries are visible on thesurface of colorless samples (FIG. 1 b). In the latter case, thedroplets are too big (˜1 μm) to cause color as a result of scattering.However, in both cases the matrix of the investigated glasses consistsof two immiscible amorphous phases: one is supposed to be silica-richand the other containing more sodium/calcium ions and phosphorus.

FT-IR reflection spectra of the two types of 45S5 glasses recorded inthe region of fundamental vibrations absorption show only subtledifferences (FIG. 2), which is not surprising owing to the integralnature of the measured spectra (averaged through a relatively largemacroscopic surface area). As compared to fused silica, where thebending vibrations of SiO₄ tetrahedral units at ˜800 cm⁻¹, asymmetricstretch modes at ˜1123 cm⁻¹ and ˜1220 cm⁻¹ are observed (FIG. 2), the45S5 glass has these optical modes shifted towards lower frequencies(˜700 cm⁻¹ for bending and ˜1050-1100 cm⁻¹ for asymmetric stretchingvibrations, respectively), which is typical for any other alkali andalkaline earth containing glasses. Also, new bands at ˜900 and ˜850 cm⁻¹emerge, which are associated with the stretching vibrations of SiO₄tetrahedra having one or two non-bridging oxygens (NBO) in the nearestsurrounding of Si atoms (so-called Q³ and Q² groups). The band at ˜600cm⁻¹ is usually attributed to phosphate complexes, having P—O bendingvibrations in this range.

In principle, viscosity of the phase separated silicate glass should behigher than the viscosity of homogeneous glass of the same composition,because the separated silica-rich matrix has high viscosity anddominates flow behavior. Indeed, the activation energy of viscous flowfor the phase separated glasses, calculated using Ozawa plot for glasstransition temperature determined at various heating rates, is found tobe dependent on the degree and type of phase separation. Thus, the valueof the activation energy of viscous flow for spinodally phase separatedglass was found to be ˜100 kJ/mol lower than the one for droplet-likephase separated 45S5 glass. We can speculate then that in the case ofdroplet-like phase separation more alkaline ions are removed from theglass matrix into the droplets, leading to higher viscosity of theresidual silica-rich phase.

The 45S5 glass transforms into a glass-ceramic by heating the materialat >700° C. for more than 0.5 h, resulting in several possiblecrystalline phases viz. Na₂Ca₂Si₃O₉, Na₂CaSi₂O₆-combeite andNa₂Ca₄(PO₄)₂SiO₄-silicorhenanite. XRD data of as-prepared glasses andfully crystallized samples (after DSC scans) are shown in FIG. 3.Predominant crystalline phase identified in both ceramics prepared fromdroplet-like and spinodally phase-separated parent glasses is combeiteNa₂CaSi₂O₆. For a spinodally phase-separated glass the additionalreflections in FIG. 3 b are caused by crystalline Si, which was added tothe powder for calibration purposes.

From comparison of typical DSC scans for the investigated samples (FIGS.4 and 5) it can be ascertained that crystallization kinetics differs forglasses with different types of phase separation and it also depends onwhether the sample is in bulk or powder form. In particular, theactivation energy of crystallization calculated according to the Ozawamethod is found to be higher for the bulk glass with droplet-like phaseseparation (compare curves for bulk samples in FIGS. 6 and 7). This canbe explained by the fact that crystallization of this glass starts at˜30 K lower temperatures than that of the spinodally phase separatedsample, where the viscosity of supercooled liquid is generally higher.The higher viscosity means more constraints for structuralrearrangements needed for crystallization to occur. In the case of glasswith spinodal type of phase separation, crystallization starts at highertemperatures where the viscosity of the supercooled liquid is lower(FIGS. 6 and 7) and structural rearrangements are easier. Accordingly,Applicants have determined that that big droplets of Na/Ca/P-rich phaseprovide more nuclei for crystallization, initiating the process at lowertemperatures.

Decrease in the particle size of powder samples causes significantbroadening of the crystallization peak and its shift towards lowertemperatures (see e.g., FIGS. 4 and 5). The activation energy forcrystallization becomes much higher than for the bulk samples and almostequal in magnitude for the droplet-like and spinodally phase-separatedglasses when the particle size is less than 150 μm (see e.g. FIGS. 6 and7). The onset temperature of crystallization also becomes almost thesame for both types of the material and simultaneously shifts by ˜30-50K towards lower values in comparison to bulk crystallization. Therefore,Applicants have determined that extended surface area of powderedsamples provides a high enough concentration of nucleation sites thatthe entire crystallization is governed by the processes on the surface.

A typical SEM picture of polycrystal formed at the surface of 45S5 BG isshown in FIG. 8. The value of the crystallization activation energy forpowdered samples of <300 μm sizes (˜350-390 kJ/mol) agrees well withactivation energy values ˜350 kJ/mol obtained earlier for 45S5 powderprepared by tape casting technique (particle sizes <10 μm).

The crystallization kinetics as studied with DSC are usually analyzedwith Johnson-Mehl-Avrami (JMA) nucleation-growth model. However, the JMAequation for non-isothermal conditions is valid only if a certain numberof criteria are satisfied: the entire nucleation process takes placeduring the early stages of the transformation, and becomes negligibleafterward; the overall crystallization rate is defined only by thetemperature and does not depend on the previous thermal history.Fundamental kinetic equations for non-isothermal crystal growth frompreexisting nuclei have been developed by Ozawa and a simple method ofkinetic analysis of DSC data for these processes has been proposed:

$\begin{matrix}{\frac{\alpha}{t} = {{{Af}(\alpha)}^{({- \frac{E_{a}}{RT}})}}} & (1)\end{matrix}$

where α is fraction of crystallized volume

$\begin{matrix}{\alpha = {\frac{1}{\Delta \; H_{c}}{\int_{0}^{T}{\varphi \ {{T}.}}}}} & (2)\end{matrix}$

Here φ is the specific heat flow measured with DSC (W/g) and ΔH_(c) isthe total enthalpy change associated with the crystallization process;the pre-exponential factor A and activation energy E_(a) are kineticparameters that should not depend on the temperature T and α; and

f(α)=m(1−α)[−ln(1−α)]^(1-1/m)  (3)

is an algebraic expression of the JMA model.

It has been demonstrated that the JMA exponent m is a characteristicparameter linked to crystal forming morphology. In particular, m˜1 meanspredominant surface crystallization, while m˜3 corresponds tothree-dimensional bulk crystallization. If one applies JMA modelstraight to the investigated 45S5 glasses, then m≈1 is typicallyobtained for powdered samples with <300 μm particle sizes and m≈3-4 forthe bulk samples. However, the validity of JMA equation for theinvestigated glasses should be demonstrated first. A simple test for theapplicability of JMA model is proposed by Malek. It is based on theanalysis of probe functions:

$\begin{matrix}{{y(\alpha)} = {\varphi \; ^{({- \frac{E_{a}}{RT}})}}} & (4) \\{{z(\alpha)} = {\varphi \; T^{2}}} & (5)\end{matrix}$

For JMA equation to be valid the maximum of the z(α) function shouldoccur around α=0.63±0.02 value. As seen from FIG. 9, the maximum forz(α) function is around α=0.55-0.56 for bulk (and powder with particlesizes >300 μm) samples of both spinodally and droplet-likephase-separated samples. Accordingly, we may conclude that the JMAequation is not applicable to describe the crystallization kinetics ofbulk 45S5 BG, which is consistent with previous studies. The same istrue for powdered samples with particle sizes <300 μm of droplet-likephase separated glass, which also demonstrate significant deviation fromα=0.63 value expected for the validity of the JMA model (FIG. 9 b); theα values are significantly higher than predicted. However, in the caseof spinodally phase-separated glass (FIG. 9 a), the JMA model can beapplied for the analysis of crystallization kinetics of powder sampleswith small particle sizes (<300 μm). Accordingly, the mean value of m(about 1) obtained for small particles (<300 μm) of spinodallyphase-separated glass is consistent with surface initiatedcrystallization mechanism proposed by Clupper and Hench for tape cast45S5 glass (particle size less than 10 μm). Therefore, Applicants havedetermined that for small particle sizes (when the crystallization ismostly surface-driven) and spinodal-like phase separation, thecrystallization kinetics can still be approximated with the JMA model.

The evolution of nanopores during phase separation and crystallizationis studied with PAL spectroscopy. The main potential of the PALspectroscopy method lies in its ability to characterize the local freevolumes (including both open and closed pores) in materials on asub-nanometer scale. The PAL method is particularly effective whenpositronium (Ps) is formed (ortho-Ps and para-Ps), because the energeticand geometric characteristics of this electron-positron bound state(hydrogen-like atom) are well determined, which allow quantification offree volume dimensions. Thus, it is possible to estimate the averagehole size from the ortho-Ps lifetime in a given material. Assumingapproximately spherical shape of the free volume, the ortho-Ps lifetime(τ_(o)) can be related to the average radius of holes (R) by asemi-empirical Tao-Eldrup equation:

$\begin{matrix}{{\tau_{o} = \lbrack {{2( {1 - \frac{R}{R + {\Delta \; R}} + {\frac{1}{2\; \pi}{\sin ( \frac{2\; \pi \; R}{R + {\Delta \; R}} )}}} )} + 0.007} \rbrack^{- 1}},} & (6)\end{matrix}$

where ΔR is an empirically determined parameter (in the classical caseΔR≈0.1656 nm), describing effective thickness of the electron layerresponsible for the pick-off annihilation of ortho-Ps in the hole.

Typical PAL spectra for 45S5 glasses and glass-ceramics are shown inFIG. 10, giving the best-fit parameters as listed in Table 2. Fourdiscrete exponentially decaying components can be distinguished in thesespectra (FIG. 10), using iterative curve fitting procedure of the LTprogram. The first component (τ₁, I₁) includes free annihilation,para-Ps decay and is related also to the positrons' bulk lifetime(τ_(b)) in the sample. The second component (τ₂, I₂) is usually causedby positrons that are trapped before annihilation in the free volumedevoid of significant electron density in the glass structure, whereformation of Ps is impossible for any other reason (geometric,energetic, inhibition of Ps formation, etc.). However, this componentmay convolute with greater bulk lifetime component of another phase, ifit is present in the material.

The remaining two components (τ₃, I₃), (τ₄, I₄) can be directlyassociated with ortho-Ps formation (Table 2). These lifetimes can berelated to corresponding pores via Tao-Eldrup relation (Eq. (6))assuming ortho-Ps is in the ground (often denoted as “1 s”) state, whichis usually satisfied at low temperatures and for relatively small pores.As seen from Table 2, the as-prepared 45S5 glasses both spinodally anddroplet-like phase-separated contain almost the same amount of voidsR₃˜1.7 Å in radius (estimated from τ₃ and I₃ values). However,spinodally phase-separated glass contains more voids of larger radiusR₄˜3.6 Å (Table 2) in comparison to the droplet-like phase-separatedmaterial (estimated by τ₄ and I₄ values).

TABLE 2 Fitting results of PAL measurements in phase separated 45S5bioglass and derived ceramics: τ₁ in ns, I_(i) in % R_(i) in {acute over(Å)}. Component fit Voids Sample τ₁ (0.005) I₁ (1) τ₂ (0.005) I₂ (1) τ₃(0.01) I₃ (0.4) τ₄ (0.05) I₄ (0.2) R₃ R₄ Spinodally phase separatedGlass 0.180 50 0.420 44 1.026 4.7 2.859 1.0 1.73 3.58 Ceramics 0.180 550.420 41 1.467 2.8 3.762 0.6 2.33 4.16 Droplet-like phase separatedGlass 0.180 47 0.420 47 1.035 4.7 3.444 0.3 1.73 3.97 Ceramics 0.180 520.420 45 1.383 2.9 4.240 0.3 2.22 4.43

These void sizes normally are associated with fine pores within theoxide building blocks (individual coordination polyhedra) of the silicanetwork and their interconnection. After droplet-like phase separationor ceramization process the voids agglomerate together, forming voids oflarger dimensions (R₃˜3.2-3.3 Å and R₄−4.2-4.4 Å in radii, see Table 2),which most probably reside within the boundaries between the devitrifiedcrystallites and residual glassy phase, or different glassy phases. Atthe same time structure densifies at a finer scale, as indicated by theincrease of I₁ intensity after ceramization of both types of 45S5phase-separated parent glasses (Table 2). This behavior can beunderstood if one takes into account the fact that bulk lifetime ofpositrons is generally lower in crystalline material (formedcrystallites) than in the corresponding glass.

The example shows that type of phase separation (spinodal vs.droplet-like) has a pronounced effect on the devitrificationcharacteristics of 45S5 BG. In particular, it appears that activationenergy of viscous flow for a spinodally phase separated 45S5 glass islower than that for the droplet-like phase separated glass.Crystallization starts at lower temperatures and the activation energyof crystallization is higher for droplet-like phase separated glass,whereas the spinodally phase separated glass crystallizes at highertemperatures and, therefore, has a lower activation energy ofcrystallization. The JMA equation is found to be not applicable to thecrystallization kinetics analysis for both types of bulk 45S5 BGs.However, for small particle sizes (<300 μm), where crystallizationprocesses are mostly surface driven, JMA equation still works undercertain conditions, viz. for spinodally phase separated 45S5 glasspowder with small particle size. The nature of phase separation alsoaffects the pore distribution at the nanoscale as shown by PALspectroscopy. As-prepared 45S5 glasses both spinodally and droplet-likephase-separated contain almost the same amount of fine voids R₃˜1.7 Å inradius, whereas spinodally phase-separated glass contains more voids oflarger dimensions R₄˜3.6 Å. This difference in sub-nanoscale structureis another possible mechanism for the difference in how the proteins andcells respond to them. After devitrification these voids show thetendency to agglomeration in both types of materials.

Example 2

The following example demonstrates the bioactivity of a glasscomposition having a spinodal distribution. In this example, 45S5bioglass is used which is characterized by its complex composition asfollows:

-   -   24.4 mol % Na₂O (sodium oxide)    -   26.9 mol % CaO (calcium oxide)    -   2.6 mol % P₂O₅ (phosphorus pentoxide)    -   46.1 mol % SiO₂ (silicon dioxide).

Referring to FIGS. 11 and 12, quenching from varying temperatures canalter 45S5 bioglass nanostructure as described above. Specifically, themelt-quench process is characterized by melting precursors at hightemperatures followed by quenching (rapid cooling) the melt to roomtemperature, producing solid bioglass.

Here, three glass “batches” were rapidly cooled from 1550, 1380, and1370° C. to room temperature. As discussed above, cooling the batches atthese different temperatures resulted in different phase separation. Thedifferences in phase separation result in varying nanostructure in,either isolated droplets (1370° C.) or spinodal interconnected (1380 &1550° C.) morphology according to SEM observations. Specifically,referring to FIG. 13, nanostructure of 45S5 glass quench at 1370° C.with droplet (a), and at 1380 & 1550° C. with spinodal nanostructures(b) observed by SEM (scale bar: 500 nm).

Each cylindrical glass column produced from the three different sampleswas cut into a set of 2 mm discs, polished to achieve an optical finish.All samples were re-cut, autoclaved, and sterilized through acetonewashes. All samples were pre-incubated in PBS for 72 hrs at 37° C. Allsamples were seeded with 30,000 cells/cm² in 3.5 cm dishes and twosamples from each batch were incubated for 2, 6, 12, and 24 hrs. Sampleswere then subsequently fixed, and stained for DAPI, A488-Phalloidin, andVinculin. All samples were then imaged for cell density, morphology, andattachment.

It should be understood that, according to current research, uponcontact with protein-rich body fluids, the surfaces of 45S5 bioglasssamples are instantaneously coated and saturated with numerousextra-cellular matric (ECM) proteins, The cells of the body, therefore,do not actually contact the biomaterial itself, but rather attach to themolecular architecture of the surface-adsorbed proteins.

Referring to FIG. 14, the effects of nanostructure on cellular densityand adhesion are shown. Specifically, in (i) and (ii), cell density wasvisualized with DAPI for the 1370° C. and 1550° C. samples,respectively, and, in (iii) and (iv), Actin was visualized withAlexa-488-Phalloidin for the 1370° C. and 1550° C. samples,respectively. MC3T3-E1 pre-osteoblasts fixed and imaged 2 hours afterseeding onto 45S5 bioactive glass samples that were prepared byquenching the melt at 1370 and 1550° C. Note significant increase incell number and attachment on the sample quenched at 1550° C.

Referring to FIG. 15, results confirm that cells are able to respond todifferences in the nanostructure of 45S5 bioglass. Average cell densityof MC3T3-E1 preosteoblasts on polished 45S5 bioglass samples prepared byquenching the melt at 1370° C., 1380° C., and 1550° C. are shown. Fromthe results above, it is evident that cells preferred 45S5 bioglasssamples exhibiting spinodal nanostructure over the samples exhibitingdroplet nanostructure. (i) 2H: We observed statistically significantdifferences between the 1370° C. and 1380° C. samples in fiveexperiments, while statistically significant differences between 1370°C. & 1550° C. samples were observed in 3 experiments; (ii & iii) 6H &12H: Statistically significant differences between the 1370° C. & 1380°C. samples and between 1370° C. & 1550° C. samples were observed once;(iv) 24H: Statistically significant differences between the 1370° C. &1380° C. samples in 2 experiments were observed, while statisticallysignificant differences between 1370° C. & 1550° C. samples wereobserved in 1 experiment.

Based on these results, a strong cellular preference for samplesexhibiting spinodal nanostructure was observed. Specifically, theresults show that cells are able to react to subtle architecturalvariations in 45S5 bioglass as cells attached to samples exhibitingspinodal nanostructure in larger numbers, consistently.

In conclusion, Applicants have discovered that a glass comprisingspinodal type nanostructure is biomedically superior to the same glasscomprising droplet type nanostructure. These two types of nanostructurescan be obtained by controlling the melt cooling process, which mayinclude the melt temperature, mold temperature, casting procedure, batchsize, subsequent heat treatment, etc., among other process parametersthat affect thermal history during glass formation. Applicants note thatwithin the broad classification of spinodal or droplet typenanostructures, significant variations of the distribution of two phasesexist, which also impact cell response. In other words, the conditionsof glass fabrication may be further optimized for improving the cellresponse, but the basic premise of superior performance of the spinodalnanostructure will remain.

What is claimed is:
 1. A glass composition prepared from the processcomprising: heating a mixture of chemicals to a melt temperature to forma melt, said melt being characterized in that quenching said melt at orabove a threshold temperature results in a spinodal phase seperation,and quenching said melt below said threshold temperature results in adroplet phase seperation; and quenching said melt at or above saidthreshold temperature in a preheated mold to form said glass compositionhaving said spinodal phase seperation.
 2. The composition of claim 1,wherein said melt temperature is about 1400 to about 1600° C.
 3. Thecomposition of claim 2, wherein said threshold temperature is no lessthan about 86% of said melt temperature
 4. The composition of claim 2,wherein said melt temperature is about 1450 to about 1550° C.
 5. Thecomposition of claim 4, wherein said threshold temperature is no lessthan about 88.5% of said melt temperature
 6. The composition of claim 5,wherein said threshold temperature is no less than about 89% of saidmelt temperature
 7. The composition of claim 1, wherein said glasscomprises 45S5 bioglass.
 8. The composition of claim 1, whereinthreshold temperature is no less than about 1375° C.
 9. The compositionof claim 8, wherein said threshold temperature ranges from 1375 to 1550°C.
 10. The composition of claim 8, wherein said threshold temperatureranges from about 1375 to about 1380° C.
 11. The composition of claim 1,wherein said preheated mold is preheated to about 100 to about 300° C.12. The composition of claim 1, further comprising calcining saidmixture before forming said melt.
 13. The composition of claim 1,further comprising cooling said mixture from said melt temperaturebefore quenching.
 14. A process of preparing a glass comprising: heatinga mixture of chemicals to a melt temperature to form a melt, said meltbeing characterized in that quenching said melt at or above a thresholdtemperature results in a spinodal phase seperation, and quenching saidmelt below said threshold temperature results in a droplet phaseseperation; and quenching said melt at or above said thresholdtemperature in a preheated mold to form said glass composition havingsaid spinodal phase seperation.
 15. The process of claim 14, whereinsaid melt temperature is about 1400 to about 1600° C.
 16. The process ofclaim 14, wherein said threshold temperature ranges from 1375° C. toabout 1550° C.
 17. A method of using a glass to promote bioactivity,said method comprising: disposing said glass composition in a cellularenvironment, said glass being prepared by a process comprising at least:heating a mixture of chemicals to a melt temperature to form a melt,said melt being characterized in that quenching said melt at or above athreshold temperature results in a spinodal phase seperation, andquenching said melt below said threshold temperature results in adroplet phase seperation; and quenching said melt at or above saidthreshold temperature in a preheated mold to form said glass compositionhaving said spinodal phase seperation; and facilitating cellularattachment and proliferation on said glass composition.
 18. The methodof claim 17, wherein said cellular proliferation is greater than that onglass having formed from the same said mixture but with said dropletphase seperation.
 19. The method of claim 17, wherein said cellularenvironment is a body of an animal.
 20. The method of claim 19, whereinfacilitating cellular attachment comprises sustaining the life of saidanimal.